CN112972664A - Device and method for preparing gel droplet monocyte vaccine from blood based on microfluidic chip - Google Patents
Device and method for preparing gel droplet monocyte vaccine from blood based on microfluidic chip Download PDFInfo
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Abstract
The invention discloses a device and a method for preparing gel droplet monocyte vaccine from blood based on a microfluidic chip. The device of the invention is divided into two areas: a separation region and an encapsulation region. The separation area utilizes the micro-column array technology, blood enters from the inlet, mononuclear cells in the blood are separated, and the mononuclear cells enter the encapsulation part along with buffer solution. The packaging region utilizes a droplet technology to prepare alginate gel droplets, separated monocytes, antigens and adjuvants are wrapped in the gel droplets, the monocytes load the antigens in the gel droplets, and the alginate gel droplet monocyte vaccine can be obtained in the chip collecting region. The invention utilizes the fluid mechanics principle and the droplet microfluidic technology to realize the direct and high-efficiency preparation of the cell tumor vaccine from blood, is suitable for the personalized treatment of cancer, and has the advantages of simple and convenient operation, low cost, high vaccine preparation efficiency, good tumor treatment and prevention effect, wide application range, strong expandability and the like.
Description
Technical Field
The invention belongs to the crossing field of a microfluidic technology and cancer immunotherapy, and particularly relates to a device and a method for preparing a gel droplet monocyte vaccine from blood based on a microfluidic chip.
Background
Since the beginning of the 20 th century, methods for eliminating tumors using the exquisite specificity of the immune system have been developed. In the past decade, several effective strategies have emerged, and thus immunotherapy is now widely recognized as an important complementary tool for treating cancer patients. Cancer vaccines are amplifying tumor-specific T cell responses by active immunization and have long been considered as a key tool for effective cancer immunotherapy. Technological advances in genomics, data science, and cancer immunotherapy now allow mapping of mutations within the genome, rational selection of vaccine targets, and production of customized therapies for individual patient tumors as needed. Thus, one of the key challenges facing the clinical application of personalized vaccines is the rapid manufacture, reasonable price, and timely delivery of individually tailored vaccines.
Microfluidic technology has great potential, may provide a solution for this because it can handle samples that are not available in large quantities (e.g., cells from a patient biopsy), is cost effective, provides a high degree of automation, and allows complex models to be built for cancer research. The process can rapidly and cheaply manufacture the chip with micron-sized defined geometric shapes, thereby making the microfluidic technology accepted by the wide research community. Reagents are injected into such devices, which can be processed and analyzed 10-6To 10-16The liquid of L, thus has greatly reduced the use amount of reagent, has improved the sensitivity, has realized the accurate unicellular measurement. In small channel sizes, the viscous forces become dominant and the water-based fluid begins to behave like a viscous fluid. The resulting laminar flow system allows precise control of injected fluids, such as a steady concentration gradient or parallel flow of miscible reagents.
By using the technologies, the microfluidic chip can simply separate DNA, protein, immune cells, extracellular vesicles or rare circulating tumor cells from blood, and then perform on-chip multi-path analysis to achieve patient classification and drug quantification according to individual needs. Blood samples can be collected periodically and the patient can be observed continuously. These microfluidic devices require very small sample volumes and are capable of creating instant snapshots of cancer as a tool for cancer risk indication, early detection, tumor classification and recurrence. Therefore, the microfluidic chip is utilized to rapidly prepare the cell vaccines of different patients carrying the self-cell antigens, and the preparation time of clinical treatment is saved. It can also be easily combined with other adjuvants for combined therapy, which is expected to bring new idea for chip-assisted personalized therapy.
Disclosure of Invention
The invention aims to provide a device and a method for preparing gel droplet monocyte vaccine from blood based on a microfluidic chip. The invention utilizes hydrodynamics and droplet microfluidic technology to realize the efficient and convenient preparation of a gel droplet monocyte vaccine from blood, and the blood, buffer solution, antigen and adjuvant are sequentially introduced into a chip to obtain the alginate gel droplet monocyte vaccine, and good tumor growth inhibition and prevention effects are obtained in a mouse (BALB/c) tumor model. The device has simple structure, does not need any complex and expensive equipment, and can realize the convenient, automatic and rapid preparation of the tumor vaccine. Therefore, the invention provides a rapid and convenient vaccine production platform, and has application value in the field of personalized immunotherapy of cancers.
The purpose of the invention is realized by the following technical scheme:
a device for preparing gel droplet monocyte vaccine from blood based on a microfluidic chip comprises a separation area and an encapsulation area. The separation area comprises a blood inlet (1), a buffer solution inlet (2), a micro-column array separation area (3) and a waste liquid collection area (4); the encapsulation region comprises an antigen and adjuvant inlet (5), an oil phase inlet (6) and a gel droplet tumour vaccine collection region (7). The blood inlet (1) and the buffer solution inlet (2) are connected with the front end of the micro-column array separation area (3) through a channel, and the waste liquid collection area (4) is connected with the tail end of the micro-column array separation area (3) through a channel; the antigen and adjuvant inlet (5), the oil phase inlet (6) and the gel droplet tumor vaccine collecting region (7) are connected through a channel; the tail end of the micro-column array separation area (3) is connected with an antigen and adjuvant channel through an outlet channel for separating mononuclear cells, the two channels form a V-shaped channel, are converged and then are vertically intersected with an oil phase channel to form a T-shaped interface channel, and the T-shaped interface channel is connected with a collection area (7) through a snake-shaped channel. The micro-column array separation area (3) is composed of a micro-column array, and micro-columns are arranged according to a certain size to form a certain offset channel. The design principle is as follows: according to the transverse lateral movement theory and the diameter of the target cell, a critical value can be calculated, and the cell diameter larger than the critical value can transversely laterally move according to the array inclination angle and enter the packaging area; cells having a diameter less than the critical value will move vertically into the waste collection region. The cells entering the encapsulation area are divided into discrete nano-liter water-in-oil droplets by the micro-fluidic droplet technology under the interaction between the fluid shear force and the surface tension through the T-shaped structural channel, and the droplets wrap the cells, the antigens and the adjuvants.
Further, the specific dimensions of the device are as follows: the chip is 40mm long and 10mm wide. Microcolumn array in the microcolumn array separation region (3): the height of the cylinders is 30 μm, the diameter Dp is 30 μm, the horizontal distance G between the two cylinders is 20 μm, the vertical distance Dy between the cylinders is 10 μm, the center offset distance Delta λ is 5 μm, a certain offset channel is formed, and the inclination angle theta is 5.7 degrees; according to the theoretical calculation, the critical diameter Dc is 9.3 μm, the cells in the blood which are larger than the critical diameter will make lateral movement, and separated, and the cells which are smaller than the critical diameter will vertically flow into the waste liquid collecting area. The V-shaped channel size of the packaging area is 150 mu m multiplied by 30 mu m, the T-shaped interface channel size is 50 mu m multiplied by 30 mu m, and the snake-shaped channel size is 150 mu m multiplied by 30 mu m.
The working principle of the device is as follows: and respectively introducing blood and a buffer solution into the chip, wherein the buffer solution is a mixed solution containing alginate and Ca-EDTA complex. Separating the mononuclear cells in the blood through a separation area, mixing the buffer solution carrying the mononuclear cells and the solution mixed with the antigen and the adjuvant at a V-shaped channel opening after the mononuclear cells enter an encapsulation area, vertically entering an oil phase channel to generate liquid drops, adding a proper amount of acetic acid in a collection area to gelatinize the liquid drops, and finally obtaining the alginate gel liquid drops. The resulting gel droplets encapsulate monocytes, antigen and adjuvant. And then, adding the alginate gel liquid drop into a water phase containing Perfluorooctanol (PFO), reducing the stability of an oil-water interface by the PFO through separating a surfactant, immediately transferring the microgel from the oil phase to the water phase once the oil-water interface is unstable, centrifuging to remove the oil phase, and re-dispersing the microgel into the water phase to obtain the monocyte vaccine loaded by the gel liquid drop antigen.
A method for preparing gel droplet monocyte vaccine from blood based on the device comprises the following steps: the blood inlet (1), the buffer solution inlet (2), the antigen and adjuvant inlet (5) and the oil phase inlet (6) of the device are respectively connected with a pump to be introduced with blood, buffer solution, antigen and adjuvant and oil phase, acetic acid with the volume ratio of 0.05-0.1% is added in the gel droplet tumor vaccine collecting region (7), the gel droplet tumor vaccine is transferred to a water phase containing 15-25% (V/V) perfluorooctanol after gelation, the centrifugation is carried out, the oil phase is discarded, the gel droplet monocyte vaccine is obtained, and the gel droplet monocyte vaccine is re-dispersed in the water phase for later use.
Wherein the buffer solution is PBS buffer solution containing 40-50mM alginate and 40-50mM Ca-EDTA complex;
the antigen is cancer cell lysate and is rich in all information of tumor cells;
the adjuvant is an immune inhibitory molecule, an antibody, a cytokine and the like;
the oil phase is organic fluoride, such as fluorocarbon oil;
the aqueous phase is PBS buffer.
The invention prepares the alginate gel droplet monocyte vaccine from blood based on the microfluidic chip, and the monocyte separated by the chip can keep the activity in the alginate gel droplet structure for a long time, quickly take the antigen and preserve the self-secreted cell connexin. Therefore, the invention has the advantages of simple operation, low cost, high vaccine preparation efficiency and good vaccine treatment and prevention effects, and can be applied to the field of personalized immunotherapy of cancers. It also has the following advantages and beneficial effects: (1) the monocytes can be cultured and survive in the gel droplets for a long time, and the monocytes in the gel droplets are quickly ingested due to the local increase of the concentration of the antigen, so that the antigen uptake time is greatly reduced, the antigen in the cells is saturated in only 30-40 minutes, and the vaccine preparation efficiency is improved. (2) Since the connexin released from monocytes is stored in the gel droplets for slow release, fewer monocytes are needed to achieve the ratio for specific T cell stimulation using conventional adoptive cellular immunotherapy.
Drawings
FIG. 1: (A) a schematic view of a microfluidic chip; (B) a chip structure diagram; (C, F) enlarging a detail; (D) a cylinder height map; (E) and (5) a structural dimension graph.
FIG. 2: (A) a bright field diagram of alginate gel droplet monocyte vaccine; (B) (ii) a fluorescent field map of alginate gel droplet monocytes; (C) monocytes take fluorescent field patterns of antigen in the droplets at different times; (D) specific cytotoxic T lymphocyte assays for gel-droplet vaccines of varying monocyte counts; (E) release assay of connexin Cx43 in gel droplets.
FIG. 3: (A) volume measurement of tumor within 29 days; (B) body weight measurements of mice over 29 days.
FIG. 4: (A) tumor volume measurement within 29 after immunization; (B) the survival rate of the mice.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: preparation of gel droplet monocyte vaccine using chip
A device for preparing gel droplet monocyte vaccine from blood based on a microfluidic chip has a structure shown in figure 1: including the separation region, encapsulation region. The separation area comprises a blood inlet (1), a buffer solution inlet (2), a micro-column array separation area (3) and a waste liquid collection area (4); the encapsulation region comprises an antigen and adjuvant inlet (5), an oil phase inlet (6) and a gel droplet tumour vaccine collection region (7). The blood inlet (1) and the buffer solution inlet (2) are connected with the front end of the micro-column array separation area (3) through a channel, and the waste liquid collection area (4) is connected with the tail end of the micro-column array separation area (3) through a channel; the antigen and adjuvant inlet (5), the oil phase inlet (6) and the gel droplet tumor vaccine collecting region (7) are connected through a channel; the tail end of the micro-column array separation area (3) is connected with an antigen and adjuvant channel through an outlet channel for separating monocytes, the two channels form a V-shaped channel, the V-shaped channel is converged and then vertically intersected with an oil phase channel to form a T-shaped interface channel (shown in figure 1F), and the T-shaped interface channel is connected with a collection area (7) through a snake-shaped channel. The micro-column array separation area (3) is composed of a micro-column array, and micro-columns are arranged according to a certain size to form a certain offset channel. The design principle is as follows: according to the transverse lateral movement theory and the diameter of the target cell, a critical value can be calculated, and the cell diameter larger than the critical value can transversely laterally move according to the array inclination angle and enter the packaging area; cells having a diameter less than the critical value will move vertically into the waste collection region.
The specific dimensions of the device are as follows: the chip is 40mm long and 10mm wide. Microcolumn array in the microcolumn array separation region (3) (fig. 1D, E): the height of the cylinders is 30 μm, the diameter Dp is 30 μm, the horizontal distance G between the two cylinders is 20 μm, the vertical distance Dy between the cylinders is 10 μm, the center offset distance Delta λ is 5 μm, a certain offset channel is formed, and the inclination angle theta is 5.7 degrees; according to the theoretical calculation, the critical diameter Dc is 9.3 μm, the cells in the blood which are larger than the critical diameter will make lateral movement, and separated, and the cells which are smaller than the critical diameter will vertically flow into the waste liquid collecting area. Packaging area, V-channel size of 150 μm 30 μm, T-interface channel size of 50 μm 30 μm (FIG. 1F), and serpentine channel size of 150 μm 30 μm.
The chip is prepared by ultraviolet sterilization after being prepared, and 1% BSA solution is introduced into the chip before preparing the cell vaccine so as to reduce the adhesion of cells and channel components. Blood was collected from the orbital venous plexus of mice and stored in a blood collection tube, to which heparin sodium was previously added as an anticoagulant. The buffer was PBS buffer containing 50mM alginate and 50mM Ca-EDTA complex. As shown in fig. 1(a), a blood inlet (1) and a buffer solution inlet (2) are connected to a syringe pump, respectively, to drive a sample at a constant flow rate simultaneously. Flow rates of Q respectivelyv1(blood) ═ 1. mu.L/min, Qv2(buffer) 5. mu.L/min, the fraction to be separated was stable (caAround 5 min) after which the antigen and adjuvant inlet (5) and the oil phase inlet (6) are driven, again using syringe pumps, to produce droplets. Wherein the flow rate of the antigen and the adjuvant (PBS buffer solution containing the antigen and the adjuvant) is 1 μ L/min, and the flow rate of the oil phase fluorocarbon oil (HFE7500, Novec 7500Engineered Fluid) is 1 μ L/min. The antigen is FITC-OVA ovalbumin with fluorescence labeling (working concentration is 5 mu g/mL), and the adjuvant is Anti-PD-1 antibody (working concentration is 0.1 mg/mL). After the generation of stable droplets, acetic acid was added to the collection port (7) at a volume ratio of 0.1%, the mixture was gelled for 2 minutes, immediately transferred to an aqueous phase containing 20% perfluorooctanol, centrifuged (1000rpm, 1min), the oil phase was discarded, washed 2 to 3 times with 1 × PBS, and the gel droplet monocyte vaccine was redispersed in the aqueous phase (1 × PBS) and left to stand for 40 minutes for later use.
The prepared gel drop monocytes were observed by inverted fluorescence microscopy, see fig. 2A-C: FIG. 2A is a bright field image of a droplet encapsulated monocyte, and FIG. 2B is a photograph of a nucleus stained with Hoechst 33342, which is seen to be purple in the fluorescent field; the antigen was taken up into the monocytes at 40min (fig. 2C), indicating that the monocytes and the antigen were encapsulated in alginate gel droplets, increasing the local concentration of the antigen, so that the time for the monocytes to take up the antigen was shortened to about 40 min. FIG. 2D is a graph of the percentage of Cytotoxic T Lymphocytes (CTL) induced by different monocyte counts. The increasing of the number of the mononuclear cells shows that the response of CTL is enhanced, and the vaccine has better anticancer effect. Cx43 connexin, an important protein for monocyte antigen presentation, fig. 2E, the amount of Cx43 secreted by monocytes stored for gel droplets, the amount of Cx43 inside the gel droplets in black, and the amount of Cx43 in the aqueous phase outside the droplets in red. Indicating that Cx43 can be better stored in the droplets, further explaining the strong CTL response of the vaccine.
Example 2: detection of alginate gel droplet monocyte vaccine for inhibiting tumor growth
24 healthy female BALB/c mice of 5-6 weeks old are taken and injected with 1 × 10 subcutaneous injections64T1 cells were randomly divided into 6 groups 7 days later, each group containing 4 cells, each of which was infused with a different formulation (200. mu.L). PBS, cancer cell lysate-TCL preparation, GD-TCL preparation, MC-TCL preparation, GD-MC-TCL preparation, and a pharmaceutically acceptable salt thereof,GD-MC-TCL + anti-PD-1 formulations. The formulation was injected every 7 days and the tumor volume and mouse body weight were strictly observed over 29 days. As shown in figure 3, the gel droplet monocyte vaccine has good tumor inhibition effect, and the anti-cancer effect is better when the gel droplet monocyte vaccine is combined with anti-PD-1. Furthermore, the vaccine had no effect on mouse body weight.
The preparation method of each preparation comprises the following steps:
(1) PBS formulation: 1 × PBS phosphate buffer, 0.01M, pH 7.4.
(2) Cancer cell lysate-TCL preparation: 4T1 cell lysate, cultured 4T1 breast cancer tumor cells, trypsinized, dissolved in phosphate solution (PBS) at a concentration equivalent to 10 per ml7The cells were then snap frozen in liquid nitrogen (5 min), thawed (37 ℃, 5 min), repeated 5 times, and then centrifuged at 2000 rpm for 10 min. The supernatant was taken, lyophilized, and then dissolved in 1 × PBS at a working concentration of 5 μ g/mL.
(3) GD-TCL formulation: the method of example 1 was used, where the blood inlet (1) and the buffer inlet (2) were fed with buffer only, and the antigen and adjuvant inlet (5) was fed with TCL (5. mu.g/mL) only, i.e., the droplets were encapsulated with TCL only.
(4) MC-TCL formulation: mononuclear cells in blood were isolated by conventional methods and counted at 1X 106And (4) respectively. Then, the mixture was dispersed in a solution containing TCL (5. mu.g/mL) and allowed to stand in a cell culture incubator for 4 hours.
(5) GD-MC-TCL formulation: the preparation was carried out by the method of example 1, wherein only TCL (5. mu.g/mL) was introduced into the antigen/adjuvant inlet (5), and the rest was unchanged.
(6) GD-MC-TCL + anti-PD-1 formulation: the preparation was carried out by the method of example 1, wherein the antigen was TCL (5. mu.g/mL), and the rest was unchanged.
Example 3: detection of alginate gel droplet monocyte vaccine for preventing tumor growth
Preparations such as PBS, GD-TCL, MC-TCL and GD-MC-TCL (see example 2 for preparation of each preparation) were subcutaneously injected into 5-6 week-old healthy female BALB/c mice once every seven days, three times, and then, 1X 10 was subcutaneously injected into the mouse species64T1 cells. Thereafter, the body weight and tumors of the mice were monitoredVolume of (c), mouse survival time. As shown in FIG. 4, the gel droplet monocyte vaccine has a good immune effect, greatly inhibits the growth of tumor, and prolongs the survival time of the mouse to 60 days.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A device for preparing gel droplet monocyte vaccine from blood based on a microfluidic chip is characterized in that: comprises a separation area and an encapsulation area; the separation area comprises a blood inlet (1), a buffer solution inlet (2), a micro-column array separation area (3) and a waste liquid collection area (4); the packaging area comprises an antigen and adjuvant inlet (5), an oil phase inlet (6) and a gel droplet tumor vaccine collecting area (7); the blood inlet (1) and the buffer solution inlet (2) are connected with the front end of the micro-column array separation area (3) through a channel, and the waste liquid collection area (4) is connected with the tail end of the micro-column array separation area (3) through a channel; the antigen and adjuvant inlet (5), the oil phase inlet (6) and the gel droplet tumor vaccine collecting region (7) are connected through a channel; the tail end of the micro-column array separation region (3) is connected with an antigen and adjuvant channel through an outlet channel for separating mononuclear cells, the two channels form a V-shaped channel, are converged and then are vertically intersected with an oil phase channel to form a T-shaped interface channel, and the T-shaped interface channel is connected with a collection region (7) through a snake-shaped channel;
wherein, the micro-column array separation area (3) is composed of a micro-column array, the micro-column array forms a shift channel to make the mononuclear cell enter the encapsulation area, and the non-mononuclear cell enters the waste liquid collection area.
2. The device for preparing the gel droplet monocyte vaccine from the blood based on the microfluidic chip of claim 1, wherein: the design principle of the micro-column array structure is as follows: calculating to obtain a critical value according to a transverse side shift theory and the diameter of the target cell, and performing transverse side shift on the cell diameter larger than the critical value according to the array inclination angle to enter an encapsulation area; cells having a diameter less than the critical value will move vertically into the waste collection region.
3. The device for preparing the gel droplet monocyte vaccine from the blood based on the microfluidic chip of claim 1, wherein: the device is 40mm long and 10mm wide.
4. The device for preparing the gel droplet monocyte vaccine from the blood based on the microfluidic chip of claim 1, wherein: the size of the micro-column array in the micro-column array separation area (3) is as follows: the height of the cylinders is 30 μm, the diameter Dp is 30 μm, the horizontal distance G between the two cylinders is 20 μm, the vertical distance Dy between the cylinders is 10 μm, the center offset distance Delta λ is 5 μm, an offset channel is formed, and the inclination angle theta is 5.7 degrees;
in the packaging area, the V-shaped channel size is 150 μm 30 μm, the T-shaped interface channel size is 50 μm 30 μm, and the serpentine channel size is 150 μm 30 μm.
5. A method for preparing gel droplet monocyte vaccine from blood based on a microfluidic chip is characterized in that: the method comprises the following steps: connecting a blood inlet (1), a buffer solution inlet (2), an antigen and adjuvant inlet (3) and an oil phase inlet (6) of the device of any one of claims 1-4 with pumps to feed blood, buffer solution, antigen, adjuvant and oil phase respectively, adding 0.05% -0.1% of acetic acid into a gel droplet tumor vaccine collecting area (7), transferring the gel droplet tumor vaccine collecting area to an aqueous phase containing 15-25% of perfluorooctanol after gelation, centrifuging, and discarding the oil phase to obtain the gel droplet monocyte vaccine.
6. The method for preparing the gel droplet monocyte vaccine from blood based on the microfluidic chip of claim 5, wherein the method comprises the following steps: the buffer solution is phosphate buffer solution containing 40-50mM of alginate and 40-50mM of Ca-EDTA complex.
7. The method for preparing the gel droplet monocyte vaccine from blood based on the microfluidic chip of claim 5, wherein the method comprises the following steps: the antigen is cancer cell lysate, and the adjuvant comprises an immunosuppressive molecule, an antibody and a cytokine.
8. The method for preparing the gel droplet monocyte vaccine from blood based on the microfluidic chip of claim 5, wherein the method comprises the following steps: the oil phase is organic fluoride.
9. The method for preparing the gel droplet monocyte vaccine from blood based on the microfluidic chip of claim 5, wherein the method comprises the following steps: the aqueous phase is PBS buffer.
10. A gel-droplet monocyte vaccine comprising: prepared by the process of any one of claims 5 to 9.
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